Extended production of nitric oxide from microencapsulated chemical reactants

ABSTRACT

Methods and compositions are provided for generating and applying long-lasting therapeutic nitric oxide (NO) gas from the reaction of water-soluble chemical reactants microencapsulated in polymer matrices. In some applications the microencapsulated reactants are introduced in an aqueous gel, and in other applications they are introduced to the area of therapy either directly or in a medical device such as a therapeutic pad or dressing. In some applications, the microencapsulated chemical precursors are maintained in close physical proximity to one another in a limited volume, and using a limited amount of solvent residing within that same volume to extract and process the chemical precursors to form NO.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority based on U.S. Provisional ApplicationSer. Nos. 61/692,328 filed Aug. 23, 2012, and 61/707,276 filed Sep. 28,2012, which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions and methods for generating andapplying nitric oxide locally.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with application of nitric oxide in medical indicationsand with compositions and methods for extended nitric oxide (NO)generation and application locally.

The biological importance of NO is well documented. See e.g. Lancaster JR. Proc Natl Acad Sci 91 (1996) 8137-41; Ignarro et al. Proc Natl AcadSci 84 (1987) 9265-69; reviewed in Bredt D S, J Cell Science 116 (2003)9-15; reviewed in Murad F, N Engl J Med 355 (2006) 2003-11. In mammals,NO is an endogenous physiological mediator of many processes in thenervous, immune and cardiovascular systems. These include vascularsmooth muscle relaxation, which results in arterial vasodilation andincreased blood flow. NO is also a neurotransmitter and has beenassociated with neuronal activity and various functions ranging fromavoidance learning to genital erection in males and females (Kim et al.,J. Nutrition 134 (2004) 28735). NO also partially mediates macrophagecytotoxicity against microbes and tumor cells. Besides mediating normalfunctions, NO is implicated in pathophysiologic states as diverse asseptic shock, hypertension, stroke, and neurodegenerative diseases.

NO has been applied pharmacologically in various forms. See Butler andFeelisch, Circulation 117 (2008) 2151-59. One must note, however, thatNO itself is highly reactive and is not chemically stable in air or inthe body. Therefore, its pharmacological applications almost invariablyinvolve its production via a chemical reaction of variousindividually-stable precursor compounds. Organic and inorganic nitratesacting as NO donors such as nitroglycerin and sodium nitroprusside havelong been used to correct NO deficient states or to regulate theactivities of many tissues. Topical applications of NO may be used tohelp wound and burn healing, hair growth, impotence, and to causevasodilatation where needed (e.g., promoting peripheral blood flow inpatients with impaired circulation due to diabetes or other conditionsand ripening of the cervix in pregnancy). Local high concentrations ofNO (eye, skin, e.g.) are tolerated. Smith et al. (U.S. Pat. No.5,519,020) describes polymeric nitric oxide sources thought to be usefulto promote healing.

In a range of topical applications, a low persistent dose of NO isdesired. NO serves as a powerful microbicide that is effective againstantibiotic-resistant bacteria. In anti-microbial and other topicalapplications, the NO needs to be maintained in contact with the skin foran extended period of time. In anti-microbial applications, thetherapeutically-effective NO dose can be small, only a few hundred partsper million (ppm) (see, for example, Ghaffari et al., Nitric OxideBiology and Chemistry 14 (2009) 21-29), but the effectiveness of the NOdepends substantially on how long the skin contact is maintained (Omerodet al., BMC Research Notes 4 (2011) 458-465).

A technology for topical release of NO is described in Seitz et al U.S.Pat. No. 6,103,275 and the co-pending application of Seitz et al (U.S.Ser. No. 13/688,511, filed Nov. 29, 2012), which are incorporated hereinby reference. However, this technology provides a topical NO dose thatlasts for less than one hour, and an alternate approach is needed toprovide the lengthy NO skin contact required for many therapeuticapplications.

SUMMARY OF THE INVENTION

Provided herein are compositions, methods and medical devices for localpharmaceutical application of therapeutically-effective amounts of NOvia a reaction of reagents, some or all of which are initially providedin microencapsulated form.

In one embodiment, a microencapsulated reagent is provided to react in agelatinous composition that contains a solution of one reagent thatreacts with the microencapsulated reagent as it is slowly released fromits microcapsules. This embodiment provides a time-released dose of NOover extended periods of time.

In another embodiment, two or more microencapsulated water-solublereagents are provided that react to form a therapeutic NO agent that istime-released over extended periods of time.

One aspect of the present disclosure is a method in which themicroparticles containing water-soluble reagents are subjected to anamount of water necessary only to moisten the surfaces of the particles.This method is particularly useful because it substantially prolongs therelease time of the reagents beyond the release time that would beobserved if only one type of microcapsule were immersed in an excess ofwater. This, in turn, prolongs the time during which the therapeuticagent is available, compared to that of a time-release composition basedupon the direct release of a single therapeutic agent from amicroencapsulation vehicle.

In one embodiment, a method is disclosed for generating extended releasetherapeutic nitric oxide including providing a mixture of two or moretypes of sub-millimeter-scale microparticles, where each type ofparticle contains only one kind of microencapsulated reactant. Themixture comprises at least one microencapsulated nitrite salt, at leastone microencapsulated acid, and at least one microencapsulated reducingagent and further providing an activating volume of water sufficient toincipiently wet the microencapsulated reactant particles, wherein theactivating volume of water is added to the microencapsulated reactantsand extended release production of NO is initiated via a nitrous acidintermediate. In one embodiment ascorbic acid serves as both the acidand the reductant.

The mixture of sub-millimeter-scale microencapsulated reactants isprovided in a wound dressing or bandage in certain embodiments. Thesub-millimeter-scale microencapsulated reactants may be provided in amoisture-proof unit dose container that is sealed until use. Whenneeded, the container is opened and the reactants administered to atissue site in need of treatment. The container may be a wound dressingor bandage or may be a container of dry powder reactants that are pouredor sprinkled on the tissue. In one embodiment, an activating volume ofsterile water is provided together with the unit dose of reactants withinstructions for administration. In certain embodiments, a plurality ofpremeasured amounts of water are provided including an activating volumeand one or more reactivating volumes with instructions to addreactivating volumes at intervals after the initial activation. Thereactants and activating/reactivating water or an aqueous gel togetherwith instructions is provided in kit form in certain embodiments.

The present NO generation method through microencapsulation of chemicalreactants is useful because it provides for the prolonged production ofan unstable compound (such as NO) from precursors that are in achemically-stable form. Multiple microencapsulated reactants can readilybe stored mixed and in contact with one another in a dry environment,and the production of NO can be initiated simply by providing a smallamount of water to the precursor mixture. Alternatively, such a mixtureof microencapsulated reactants can be applied directly to a wound,wherein the wound environment itself provides sufficient water to causerelease of therapeutic amounts of NO. A further advantage is that thevolume occupied by the reagents and water is relatively small, promotingincorporation of this invention into dimensionally-limited objects. Suchobjects would include wound dressings, bandages, and otherphysically-thin medical articles and also physically-small medicalarticles such as vascular and other stents, catheters, pacemakers,defibrillators, heart assist devices, artificial valves, electrodes,orthopedic screws and pins and other medical articles routinelyimplanted or inserted into the body.

In an alternative embodiment, microencapsulated nitrite is mixed withactivating reactants that are in gel form. The gel slows the interactionbetween the nitrite and the activating reagents resulting in higherlevels of dissolved NO as well as prolonged NO release. The materialsand methods disclosed in this embodiment result in an increase theamount of NO produced over a several-hour period, compared to the amountthat would be observed when two or more types of particles individuallycontaining appropriate reagents are immersed together in an excess ofwater or water-based liquids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-sectional view of an embodiment of a pad containing amixture of microencapsulated reagents that react to produce NO. FIG. 1Bshows a cross-sectional view of a pad incorporating an internal elementto keep the microparticles in place. FIG. 1C is a cross-sectional viewof an embodiment of an absorbent bed-pad incorporating microencapsulatedreagents that react to produce NO.

FIG. 2 shows the NO release when 10 mg of particles comprising NaNO₂microencapsulated with zein (a plant protein) are introduced to an acidsolution of ascorbic acid as described in EXAMPLE 1.

FIG. 3 shows the arrangement of amiNO-700 probe and microencapsulatedpowder mix used in EXAMPLE 3.

FIG. 4 shows the NO release when 10 mg of particles comprising ascorbicacid microencapsulated in an ethyl cellulose matrix and 10 mg ofparticles comprising NaNO₂ microencapsulated in an ethyl cellulosematrix are incipiently wet as discussed in EXAMPLE 3.

FIG. 5 shows the NO release when 10 mg of particles comprising ascorbicacid microencapsulated in a zein matrix and 10 mg of particlescomprising NaNO₂ microencapsulated in a zein matrix are formed into awet paste with 200 microliters of water as discussed in EXAMPLE 4. Thepaste is formed by adding water to the mixture of particles at the timedesignated zero (0).

FIG. 6 shows data from EXAMPLE 5, which are the measured NOconcentrations in the first lower-HEC-concentration (circles) and secondhigher-HEC-concentration (crosses) solutions, subsequent to theintroduction of ten milligrams (10 mg) of NaNO₂ microparticles of thetype used in EXAMPLE 1. The HEC concentrations in the first and secondsolutions are 0.73 g/100 ml and 2.18 g/100 ml, respectively. The peakmeasured NO concentrations in the first and second solutions are 0.022micromoles/liter and 0.324 micromoles/liter, respectively. For bothcurves shown, the microencapsulated sodium nitrite is introduced at thetime designated zero (0).

DETAILED DESCRIPTION

Provided herein are methods, apparatus and compositions that deliverlong-lasting dosage of NO in topical applications by microencapsulatingNO producing reactants. In one embodiment the microencapsulation vehicleis a polymer matrix. The reagents and matrix together are incorporatedin sub-millimeter-scale structures that have at least one dimension lessthan a millimeter. Such structures can be particles, fibers or films.

One pharmaceutically-acceptable way of producing NO employed hereinrelies on the chemistry of nitrous acid (HNO₂). Nitrous acid is producedfrom inorganic nitrites on treatment with acids (HA) according toequation (1) below. Nitrous acid is stable in aqueous solution at lowtemperature, but it decomposes into NO and NO₂ readily at roomtemperature according to the equation (2).

In the presence of a reducing agent (such as ascorbic acid, Asc(OH)₂),the NO₂ is readily converted to NO as shown in equation (3) below.

2HA+2NaNO₂→2HNO₂+2NaA   (1),

where HA is an organic acid

2HNO₂→NO+NO₂+H₂O   (2),

nitrous acid decomposes generating nitrogen dioxide

NO+NO₂+H₂O+Asc(OH)₂→2NO+2H₂O+AscO₂   (3),

the ascorbic acid reacts to remove the nitrogen dioxide

In one embodiment of this invention, a microencapsulated reagent ismixed with a composition that provides an appropriate environment forrelease and reaction of that reagent to produce NO over an extendedperiod of time. When the microencapsulated materials come into contactwith water (or an aqueous solution), the liquid slowly penetrates theparticles and NO-producing reactants are liberated slowly over time. Thereactants then take part in reactions that produce NO.

One such embodiment of the invention utilizes a microencapsulatednitrite salt and an aqueous acidified gel with sufficient acidity toconvert the nitrite salt to nitric oxide. A reductant to help retain thenitric oxide in bioactive form is preferably included in the gel. Theacidifying agent is preferably an organic acid such as citric acid,although inorganic acids such as boric acid, for example may also besuitable. Other acidifying agents may include lactic acid, glycericacid, formic acid or other organic acids known to those of skill in theart. Inorganic acids with the appropriate pKa values can also be used ifthey are biologically acceptable (e.g. the aforementioned boric acid).The gel acidifying agent may also be a reductant, such as ascorbic acid(vitamin C) or an ascorbic acid derivative including but not limited to,3-O-ethyl ascorbic acid, other 3-alkyl ascorbic acids,6-O-octanoyl-ascorbic acid, 6-O-dodecanoyl-ascorbic acid,6-O-tetradecanoyl-ascorbic acid, 6-O-octadecanoyl-ascorbic acid, and6-O-dodecanedioyl-ascorbic acid. The preferred reductant is one havingthe reductive capability of preventing or slowing the oxidation ofnitric oxide to nitrogen dioxide and also having the capability ofdirectly reducing NO₂ to NO so that the gas released by the compositionis predominantly NO. Preferred reductants include ascorbic acid,ascorbic acid derivatives, ascorbate salts, tocopherol, erythrobates oralpha-tocopherol. Gelling agents include substances such ashydroxymethyl cellulose, hydroxyethyl cellulose, gelatin, agar, naturalgums, starches and pectins.

The medium for dissolution of the acid and reductant may be an aqueousmedium or a nonaqueous medium. Aqueous media are generally preferred andreadily prepared as gels. The acidic gel composition may additionallycontain the conjugate base of one or more of the acids used. While thebase is preferably the conjugate base of the acid used, but can beanother organic or inorganic base known to those of skill in the art.This embodiment of the invention may be applied directly to the skinstimulate circulation, to wounds to speed their healing, to the scalpand maintained there for a period of time as a treatment to stimulatehair growth, and it may be applied in any other application where localrelease of NO is beneficial.

Another embodiment of this invention is a kit for delivering anacidified gel and a microencapsulated nitrite salt. The acidified geland the microencapsulated nitrite salt are individually packaged inmoisture-proof packages, which are opened and their contents mixedtogether immediately prior to application of the mixture. In analternative embodiment, the microencapsulated nitrite salt and theacidifying agents are packaged either together or individually inmoisture-proof packages. The packages are opened and their contents aremixed with a measured amount of water or a pH-neutral aqueous gel priorto application of the mixture.

Another embodiment utilizes a mixture of two or more types ofmicroparticles, where each type of particle comprises only one kind ofmicroencapsulated reagent. The particles in the mixture collectivelycontain reagents that react with one another to produce NO. Forinstance, one type of particle in the mixture could contain sodiumnitrite and another type could contain ascorbic acid, which whenreleased together in solution will react to produce NO. The particles inwhich the reagents are microencapsulated are maintained in closephysical proximity to one another in a limited volume, and a limitedamount of water residing within that same volume is used to extract andprocess the reagents to produce the therapeutic agent.

By limited amount of water it is meant sufficient water to incipientlywet the dry microencapsulated reactant particles thereby permitting thereactants to interact and chemically react. Incipient wetness means avolume of the liquid sufficient to wet the reactant particles withoutexcess liquid present after addition of the liquid to the dry reactantparticles.

One embodiment of the present invention utilizes a microencapsulatednitrite salt, a microencapsulated acid, and a microencapsulated reducingagent. Various nitrite salts may be used, most commonly inorganic ones,such as sodium nitrite, although potassium nitrite, calcium nitrite, orany alkali metal nitrite or alkaline earth nitrite are usable. Forcertain indications, the reducing agent is selected to have thereductive capability of preventing or slowing the oxidation of nitricoxide (NO) to nitrogen dioxide (NO₂), and also having the capability ofdirectly reducing NO₂ to NO so that the gas released by the compositionis predominantly NO. Suitable reductants include ascorbic acid,ascorbate salts, tocopherols (including particularly alpha tocopherol),erythrobates, carotenoids, tocotrienols and thiols.

Citric acid is one acceptable organic acid. Other acids may includelactic acid, glyceric acid, formic acid or other organic acids known tothose of skill in the art. Inorganic acids with the appropriate pKavalues can also be used if they are biologically acceptable (e.g. boricacid). In an embodiment providing ascorbic acid as the acid, theascorbic acid serves both as the organic acid and the reducing agent.Ascorbic acid (vitamin C) is one biocompatible reducing agent fornitrites.

One production method for microencapsulation is spray-drying of a meltor polymer solution of one of the reagents to produce a finely-dividedpowder of individual particles comprising that reagent dispersed withina polymer matrix. Other microencapsulation methods such as pan coating,air suspension coating, centrifugal extrusion, fiber spinning, fiberextrusion, nozzle vibration, ionotropic gelation, coacervation phaseseparation, interfacial cross-linking, in-situ polymerization and matrixpolymerization may also be used.

For purposes of the medical indications disclosed herein, theencapsulation polymer is a biocompatible polymer. Suitable polymersinclude ethyl cellulose, natural polymers such as zein (a prolamine seedstorage protein found in certain grass species including maize andcorn), chitosan, hyaluronic acid, and alginic acid, or biodegradablepolyesters, polyanhydrides, poly (ortho esters), polyphosphazenes, orpolysaccharides (see, Park et al., Molecules 10 (2005) 141-161).

Compositions in which one chemical is microencapsulated as indicatedabove are well-known for delivery of pharmaceutical agents. See Shalabyand Jamiolkowski, U.S. Pat. No. 4,130,639; Buchholz and Meduski, U.S.Pat. No. 6,491,748. However, in virtually all of such compositions, itis the therapeutic agent itself that is microencapsulated, and thetherapeutic agent is not produced by a reaction of microencapsulatedreagents. Nitric oxide releasing polymers have been described formedical articles that involve NO adducts/donors. See e.g. Arnold, U.S.Pat. No. 7,829,553 (carbon-based diazeniumdiolates attached tohydrophobic polymers); Knapp U.S. Pat. No. 7,135,189 (a nitrosothiolprecursor and a nitric oxide donor).

Applications of embodiments of the present invention include directapplication of the microencapsulated reactants to wounds, dressings forwounds, surgical dressings, bed-pads for patients who have (or mightdevelop) bedsores, socks and other garments for diabetics and otherpatients with circulatory impairment, and orthopedic casts, as well asfor local delivery of NO for use as a vasodilator in the treatment ofsexual dysfunction. The invention also meets a need in which one desiresto have a small but long-lasting dose of NO associated with medicalarticles routinely implanted or inserted into the body such as vascularand other stents, catheters, pacemakers, defibrillators, heart assistdevices, artificial valves, electrodes, and orthopedic screws and pins.

The invention can be packaged in a wound dressing or bandage so that apart of the dressing holds a mixture of particles that contain onemicroencapsulated reagent with other particles that contain anothermicroencapsulated reagent. This part of the dressing also incorporates amaterial having water retention properties that enable maintenance ofthe appropriate amount of moisture to maintain the particles in a wetenvironment. Wetting the dressing initiates the reaction of thereagents, and the dressing begins to release NO. The dressing isstructured so that the NO is released near the wound.

One embodiment of the present invention provides for extended release ofNO from a layered pad that has multiple applications. It is shown incross-section in FIG. 1A. A mixture of two or more types ofmicroparticles 1 is contained between two layers 2 and 3, at least oneof which is a bodyside-facing layer that will transmit gaseous NO, andat least one of which is an outward-facing impermeable layer or hasmoisture-retention properties that permit transmission of appliedliquids to the microparticles inside and/or maintenance of themicroparticles in a moist environment. In applications where it isdesirable to retain NO on one side of the pad, one of the layers 2 or 3is impermeable to NO. In the mixture of two or more types ofsub-millimeter-scale microparticles, each type of particle comprisesonly one kind of microencapsulated reactant. The combination ofreactants evolved from the particles in an aqueous environment producesNO. When water is introduced to the pad, the reactants begin to bereleased, and NO production begins.

In certain embodiments, the outer layers may be separated by a spacerlayer 4 shown in FIG. 1B, which serves to maintain the spacing betweenthe outer layers and also keeps the layer of microparticles in place.The reagent-containing microparticles may be embedded in or otherwiseaffixed to the spacer layer 4 or to an inner surface of either of theouter layers 2 or 3.

Pads of the type shown schematically in FIG. 1 can be prepared to anyprescribed size and shape. The vertical dimensions of FIGS. 1A-C are notto scale, and the water absorbing material 5 may be much thicker thanthe reagent-containing pad.

Such pads have multiple applications. They can be applied to a woundsimply by placing the pad on the wound and affixing it by a covering ofa suitable layer of adhesive surgical tape. They may be incorporated ina pre-manufactured bandage or dressing. Alternatively, the bandage ordressing may be configured with a pouch that contains microencapsulatedreagents that react to produce NO. The reagents additionally can beaffixed to different layers of material, which are then assembledtogether to form the completed bandage or dressing.

Other configurations of the pad shown in FIG. 1 can serve as along-lasting antimicrobial wipe cloth. The pads may be adapted anddimensioned as inserts for garments such as socks or leggings forpatients with circulatory impairment. With an appropriate treatment ofthe edges of the material, and structure of the pad to contain themicroparticles, the pad itself can also serve as the fabric for socks,gloves and other garments for patients with circulatory impairment.These garments may be activated by the moisture naturally available fromthe patients' skins or water may be added for activation.

Another embodiment of this invention is a bed-pad shown in FIG. 1C thatcomprises the microparticle-containing pad described above with anabsorbent or permeable layer 5 and an impermeable layer 6 situatedbeneath it. This absorbent bed-pad is appropriate for patients who haveor are beginning to develop decubitus ulcers (bedsores). Such patientsproduce modest amounts of moisture through incontinence andperspiration. The moisture will activate the NO producing pad, andexcess moisture will be absorbed by the layer beneath the pad. Thisarrangement will have the effect of bathing the bedsore in NO which willstimulate healing and prevent further enlargement of the ulcerated area.

In a different application, small doses of NO, topically applied to thepenis, have been shown to be very effective in rapid stimulation ofpenile erection in male rats (Han et al., Journal of Sexual Medicine 7(2010) 224). The present invention provides such a topical applicationof NO for similar effect in humans. Present systemic drugs for sexualdysfunction have multiple negative side effects and take some timebefore becoming effective. A fast-acting, topical treatment is highlydesirable in terms of its controllability and anticipated lack ofsystemic side effects. The NO generating reactants may be provided as adry coating on dressings that are adapted for placement on erectiletissue. One example would be the interior of dressings such as male orfemale condoms. Wetting of the dressing for application to the erectiletissue activates the reactants that then release NO over an extendedperiod of time.

Another embodiment of this invention is a condom having the innersurface coated with a coating comprising a mixture ofindividually-microencapsulated reagents which, when in aqueous solution,react together to produce NO. The microparticle size range for thisembodiment is between 0.01 and 100 microns with the range of 1 to 10microns being preferred. The smaller particles facilitate theirpreparation in a coating that is adherent to the inner surface of thecondom and also provide a NO release on a time scale of minutes insteadof hours. In employing one such embodiment, the user would apply anaqueous compound such as K-Y Jelly (manufactured by McNEIL-PPC, Inc.,Ft. Washington, Pa.) to the erectile tissue prior to putting on thecondom. As the microparticles come into contact with the aqueouscompound, NO release begins. The NO released is contained by the condomuntil it is absorbed transdermally into the erectile tissue, stimulatingand prolonging its erection.

Another embodiment of this invention is a sexual arousal gel kitcomprising a container of an aqueous gel compound similar to K-Y Jellyand a moisture-proof package of a mixture ofindividually-microencapsulated reagents which, when in aqueous solution,react together to produce NO. The package is opened and mixed with theaqueous gel prior to use, and applied to the external genitalia of maleand/or female users to stimulate blood flow therein, thereby promotingpenile and clitoral erections. Such a kit is useful for treatment ofsexual dysfunction and for enhancing sexual satisfaction in males andfemales.

While clinical studies with humans have not been performed, studies withrats have suggested that the NO gel composition described by Seitz etal. (U.S. Pat. No. 6,103,275) stimulates hair growth. It is known thattopical vasodilators such as Minoxidil can be effective in mitigatinghair loss and stimulating hair growth in humans, and it is likely thattopical application of long-lasting low dose NO, which is a potentvasodilator, will have a therapeutic effect for hair loss. Thus anotherapplication of the extended release formulations disclosed herein liesin devices and compositions that mitigate hair loss and stimulate hairregrowth. One particular embodiment is a skull-cap comprising thematerial shown in FIG. 1 for use in treatment of hair loss. The cap ismade to fit over the balding areas of the patient's head, is applied andmoistened with water to activate it.

EXAMPLE 1 Demonstration of Time-Release of Reagent from One Type ofParticle

Microparticles having 10% by weight sodium nitrite (NaNO₂) in a zeinmatrix were prepared by spray drying a solution of sodium nitrite, zeinand a volatile solvent. Zein is a proline-rich protein obtainable fromcorn that can be used in processed foods and pharmaceuticals as acoating and encapsulation matrix agent. It is classified as generallyrecognized as safe (GRAS) by the U.S. Food and Drug Administration. Thesolution was 10% zein (Flo Chemicals, 29 Puffer St., Ashburnham, Mass.01430 (Lot F40000111C6)) dispersed in a mixture of 90:10 ethanol:water.The solution was dispersed into the dryer using a spinning diskatomizer. The microparticles formed in this way had diameters rangedbetween 10 and 100 microns and included a matrix of zein throughoutwhich sodium nitrite is distributed. The zein is not water soluble, andwhen the microparticles are exposed to water, the water slowly diffusesinto the zein matrix, dissolves the NaNO₂, and a solution containingNaNO₂ slowly diffuses back out of the particle, thereby producing arelease of the NaNO₂ over a substantial period of time.

One hundred milliliters (100 ml) of water solution containing 5.6 gcitric acid, 5.2 g of ascorbic acid and 0.3 g of PE9010 (a preservativemanufactured by Schülke and Mayr, 30 Two Bridges Road Suite 225,Fairfield, N.J. 07004, USA) was prepared. Forty milliliters (40 ml) ofthis solution was placed in a beaker. The NO concentration in thesolution was monitored with an inNO-T nitric oxide measuring system withan amiNO-700 probe (Innovative Instruments, Inc., Tampa, Fla. 33637).Ten milligrams (10 mg) of microparticles comprising sodium nitritemicroencapsulated with zein were added to the solution at the timedesignated zero (0) and the NO content of the solution was recordedafter to their addition, with the result being shown in FIG. 2. Thecurve of FIG. 2 shows that the NO production initiated when themicroparticles were added, rose quickly to a peak and then diminishedsignificantly during a 3-hour period. NO that is produced evolves fromthe liquid on a time scale of about 20 minutes, so the measured NOprofiles are characteristic of the production rate of NO from thereaction of sodium nitrite emanating from the microencapsulatedparticles with the acids in the liquid according to reactions (1)-(3)above.

EXAMPLE 2 Demonstration of Minimal NO Production when MicroencapsulatedReagents are Exposed to an Excess of Water

Microparticles having 10% ascorbic acid in a zein matrix were preparedby spray drying a solution of ascorbic acid, zein and a volatilesolvent. The microparticle diameters ranged between 10 and 100 microns.Ten milligrams (10 mg) of these microparticles were mixed with 10 mg ofmicroparticles of the type used in EXAMPLE 1. The mixture was added to abeaker containing 40 ml deionized water, which was continuously stirred.The NO content of the water was monitored with the same apparatus usedin EXAMPLE 1. During three hours of monitoring, no detectible NO wasobserved.

EXAMPLE 3 Demonstration of Long-Lived NO Production whenMicroencapsulated Reagents are Incipiently Wet

Microparticles having 10% by weight sodium nitrite in an ethyl cellulosematrix were prepared by spray drying a solution of sodium nitrite, ethylcellulose and a volatile solvent. The solution was 12.5% ethyl cellulose(Ethocel Standard 7 from Dow Chemicals (Lot U105013T01)) dispersed inethanol. The solution was dispersed into the dryer using a spinning diskatomizer. The microparticle diameters ranged between 10 and 100 microns.Additionally, microparticles having 10% by weight ascorbic acid in anethyl cellulose matrix were prepared by spray drying a solution ofascorbic acid, ethyl cellulose and a volatile solvent. The solution was12.5% ethyl cellulose (Ethocel Standard 7 from Dow Chemicals (LotU105013T01)) dispersed in ethanol. The solution was dispersed into thedryer using a spinning disk atomizer. These microparticle diameters alsoranged between 10 and 100 microns.

Ten mg of each type of particle was mixed together and placed on pieceof weighing paper that contained the powder in a small fold in thepaper. The amino-700 probe tip was inserted into and completely coveredby the powder mix as shown in FIG. 3. Two hundred microliters (200 μl)of deionized water was used to wet the powder mixture (at the timelabeled “1” on FIG. 4), and the excess water was absorbed by the paper.Thus the particles remained in a state of incipient wetness where waterresided primarily on their surfaces and in narrow interstices betweenthe particles. The NO signal was recorded for the next five and one-halfhours. That recording in FIG. 4 shows that NO production rose during thefirst half hour, and then decreased very slowly for the next four hours.Abruptly, after a total time of four and one-half hours, the NO signaldropped linearly toward zero. The drop in signal is concluded to be dueto complete evaporation of the water in the sample. An additional onehundred microliters (100 μl) was added after the signal dropped (at thetime labeled “2” in FIG. 4, and the signal again increased, confirmingthis conclusion. This result is surprising because of the longevity ofthe NO release compared to that observed in EXAMPLE 1.

EXAMPLE 4 Demonstration of Long-Lived NO Production withMicroencapsulated Reagents in a Moist Paste

Ten milligrams (10 mg) each of the particles used in EXAMPLES 1 and 2(i.e. particles comprising 10% by weight sodium nitrite encapsulated ina zein matrix and particles comprising 10% by weight ascorbic acid in azein matrix) were mixed together and placed in a small plastichalf-cylinder. The amino-700 probe tip was completely covered by theparticle mix in an arrangement similar to that used in EXAMPLE 3 andshown in FIG. 3. A volume of 200 μl of deionized water was used to wetthe mixture to a consistency of a moist paste, and the NO signal wasrecorded for the next eight hours.

That recording is shown in FIG. 5. NO production rose slowly for thefirst hour, and then remained relatively constant for at least theremaining seven hours, at which time the experiment was discontinued.This result is surprising in terms of the constancy and longevity of theNO release in comparison to the result of EXAMPLE 1.

First, it was noted there is a significant difference between the amountof NO measured in EXAMPLE 2 (where there was not an observable amount ofNO released) and EXAMPLES 3 and 4 (substantial NO release), even thoughthe same weights of reagent were used in both experiments. Thisdifference is surprising, but without being limited by theory, one mayconsider several possibilities as to why this is the case. Theenvironment for the NO production process in EXAMPLE 2 is substantiallydifferent from that in EXAMPLES 3 and 4, and these differences highlightsome of the aspects of the present invention.

NO is unstable in the air and in vivo and its use as a therapeutic agentrequires that it be produced by reaction of chemical precursors whichare individually stable. In EXAMPLES 3 and 4, the reservoirs of thechemical precursors (e.g. the particles in which the reagents aremicroencapsulated) are maintained in close physical proximity to oneanother in a limited volume, and a limited amount of solvent residingwithin that same volume is used to extract and process the reagents toform NO. To promote a long-duration time release, these chemicalprecursors are also microencapsulated as described above. EXAMPLE 2 ischaracteristic of what would happen if one sought to produce atime-released dose of NO simply by introduction of two microencapsulatedreagents into a water-based pharmaceutical preparation having asignificant volume or simply introduced them into the bloodstream or thealimentary tract. The reagents that evolve from the encapsulationvehicles are quickly diluted, and their rate of reaction with each otherwill be slowed very significantly. Additionally, in the particular caseof production of NO through reactions (1)-(3), it is also known that thereaction rate is pH sensitive, and decreases to zero as the pHapproaches a neutral pH of 7. In EXAMPLE 2, the only agent that lowersthe pH of the solution is the ascorbic acid evolving from themicroencapsulation vehicle particles. When the ascorbic acid is verydilute, it cannot bring the pH of the entire solution to a value that islow enough to allow the reaction producing NO to proceed at anysignificant rate. However, in the cases of EXAMPLES 3 and 4 (which areembodiments of the present invention), the reagents are being dispensedfrom particles that are in close proximity to one another, and theamount of solvent into which the reagents are dispensed is very small.Therefore the local reagent concentrations are orders of magnitudehigher than those appropriate to EXAMPLE 2. The reaction to produce NOcan proceed more rapidly, both because of the high reagentconcentrations, and because the much higher local concentration ofascorbic acid substantially decreases the pH so that the reaction canproceed at a greater rate, producing more NO per unit time.

A further surprising result is the marked contrast between the timedependence of NO production in EXAMPLE 1 (wherein the NO productiondiminishes very substantially within three hours) and that observed inEXAMPLE 3 (where the NO production continues for about five hours) andEXAMPLE 4 (where the NO production continues at a relatively constantlevel for at least 8 hours).

In EXAMPLE 1, particles containing microencapsulated NaNO₂ areintroduced to an acid solution comprising citric and ascorbic acids.Immediately upon introduction of these particles, the solution begins todiffuse into the particles and to dissolve the encapsulated NaNO₂.Subsequently, the reactions (1)-(3) producing NO take place eitherwithin the particle itself, or after dissolved NaNO₂ diffuses out of theparticle into the acid solution. This scenario is characteristic of theusual application of microencapsulated time-release compositions whereinan active component is microencapsulated and is subsequently introducedinto an environment in which that active component is released from themicroencapsulation vehicle, and the active component becomes a minorconstituent of that environment. Generally, the release rate of themicroencapsulated substance decreases with time as the supply ofmicroencapsulated material diminishes within the microencapsulationvehicle. This characteristic decrease is seen in FIG. 2, which shows thetime-dependence of NO production in EXAMPLE 1.

While the NO production in EXAMPLE 1 decreases substantially over athree hour period, the NO production in EXAMPLES 3 and 4 persists at afairly constant rate for at least seven hours. The difference issurprising and is particularly useful in some applications such asdressings for wounds, because one desires to keep the wound bathed in alow concentration of NO to promote healing and provide protectionagainst bacteria. Without being limited by theory, it is believed thatthe explanation for this useful modification of the time-releasecharacteristic again lies in the placement of the reservoirs of thechemical precursors (e.g. the particles in which the reagents aremicroencapsulated) together in close physical proximity to one anotherwithin a limited volume, and use of a limited amount of solvent residingwithin that same volume to extract and process the reagents to form NO.In this configuration, the kinetics of the process of extraction andreaction of the microencapsulated reagents is likely to be more complexthan it is in the case of EXAMPLE 1, which represents the traditionalapplication of delayed time release of a microencapsulated agent. InEXAMPLES 3 and 4, where the extraction of microencapsulated reagentsfrom the sub-millimeter-scale particles takes place with a minisculeamount of water, the concentrations of the reagents at the surface ofthe particles is much higher. It is possible that these local elevatedconcentrations modify the dynamics of diffusion of the reagents out ofthe particles, slowing the diffusion because the reagent concentrationgradient between the inside and outside of the particle is smaller.

Additionally, with the high concentrations of reagents present, itbecomes more likely that one dissolved reagent will diffuse into aparticle containing another reagent with which it reacts. To the extentthat this occurs, gaseous NO may be produced within the particle. Theproduction of a gas within the channels where diffusion is taking placeis likely to slow the diffusion of liquids in those same channels whichis the essential to bring the reagents out of the particles within whichthey are microencapsulated. Thus the total NO production rate may bediminished because a small amount of NO being formed inside theparticles.

EXAMPLE 5 Demonstration of Long Time-Release of NO from aMicroencapsulated Reagent in the Presence of Different Concentrations ofGelling Agent

A first lower viscosity gel-form acid solution was prepared as follows:0.73 grams of hydroxyethyl cellulose (HEC), 5.6 g citric acid, 5.2 g ofascorbic acid, and 0.3 g of PE9010 was mixed with water to make up 100ml. A second higher viscosity gel form acid solution was prepared asfollows: 2.18 grams of hydroxyethyl cellulose (HEC), 5.6 g citric acid,5.2 g of ascorbic acid, and 0.3 g of PE9010 was mixed with water to makeup 100 ml. The only difference between these solutions and that used inEXAMPLE 1 was the presence of the gelling agent, HEC, which is awater-soluble polymer derived from cellulose. The HEC used in thisexample had a mean molecular weight of 750,000, and when the HEC ismixed in, the solution becomes a high-viscosity gel. Both the solutionsprepared with HEC have an increased viscosity compared to the solutionused in EXAMPLE 1. Both the gel solutions are easily stirred withlaboratory stirring apparatus, but the higher-concentration-HEC solutionis considerably more viscous.

Twenty-five milliliters (25 ml) of the first solution prepared wasplaced in a beaker and continuously stirred. The NO concentration in thesolution gel was monitored with an inNO-T nitric oxide measuring systemwith an amiNO-700 probe. Ten milligrams (10 mg) of the NaNO₂microparticles of the type used in EXAMPLE 1 were introduced to the gelsolution and the NO concentration in the gel solution was recorded forthree hours. When the microparticles were introduced to the gelsolution, production of small bubbles around the microparticles wasobserved.

Twenty-five milliliters (25 ml) of the second solution prepared wasplaced in a beaker and continuously stirred. The NO concentration in thesolution gel was monitored with an inNO-T nitric oxide measuring systemwith an amiNO-700 probe. Ten milligrams (10 mg) of the NaNO₂microparticles of the type used in EXAMPLE 1 were introduced to the gelsolution and the NO concentration in the gel solution was recorded foreight hours. When the microparticles were introduced to the gelsolution, bubble production was observed to be much less pronounced andto take place over a shorter period of time than was observed whenintroducing the microparticles to the first solution.

FIG. 6 shows a comparison of the measured NO concentrations in the firstlower-viscosity (circles) and second higher-viscosity (crosses)solutions, subsequent to the introduction of ten milligrams (10 mg) ofNaNO₂ microparticles of the type used in EXAMPLE 1. The lifetime of theNO release in the solution with the higher concentration of HEC ismarkedly longer. The NO concentration in the second solution (containing2.18 g/100 ml HEC) reaches a peak that is more than an order ofmagnitude higher than that reached in the first solution and decays tohalf its peak value in about eight hours, while the NO concentration inthe first solution (containing only 0.73 g/100 ml HEC) decayed to halfits peak value in about one hour. The difference is dramatic. The NOreleased in the reaction of the initially-microencapsulated reagent istransferred much more effectively to the solution when HEC is present atthe higher concentration. Both the long duration and high concentrationof the NO release to the gel solution with a high HEC concentration aresurprising and useful in therapeutic applications where a long-duration,therapeutically-effective dose of NO is desired.

Without being bound by theory there are two effects to consider inunderstanding the difference between the evolution of the NOconcentration in the solutions with the high and low concentrations ofthe gelling agent, HEC. These effects are the kinetics of the removal ofthe NaNO₂ from the microparticles and the transfer of NO formed into theliquid.

The kinetics of the removal of NaNO₂ from the microparticles depends onboth the diffusion of water into the particles and the diffusion ofsolution containing dissolved NaNO₂ back out of the particles. Thediffusive flow of solution within the particle takes place inirregularities in the particle that provide channels for the solutionflow. HEC is a large, water-soluble polymer molecule, and when it ispresent in the solution, it substantially increases the viscosity of thesolution and inhibits diffusive flow of the solution within the channelsin the microparticle. Therefore the release of NaNO₂ to the solutionoutside the particle is substantially slowed. This slowing of thediffusion in and out of the microparticle is anticipated to beproportional to the HEC concentration; and such a slowing of diffusionis consistent with the reduction observed in the amount of bubblesformed around the particles when they are introduced to thehigh-HEC-concentration solution.

When the NaNO₂-rich solution finally does diffuse out of themicroparticle, the NaNO₂ reacts rapidly in the acid environment there toproduce NO. NO dissolves fairly slowly into the ambient liquid, and ifthe NO production rate is relatively high, NO-filled bubbles will formon the surface of the microparticles, and when they become large enough,they will migrate into the liquid. Once the bubble becomes free-floatingin the liquid, it will travel to the surface of the liquid, pop, anddisperse the NO into the air above the liquid. During the process ofbubble formation and transport through the liquid, the NO is alsodissolving into the liquid. If the bubble formation process is rapid,the bubbles reside on the surfaces of the microparticles for a shorttime, they are rapidly released into the liquid. If the bubbles risethrough the liquid rapidly, they effectively transport a substantialfraction of the NO out of the liquid, and it is never dissolved.

HEC, in proportion to its concentration, slows the diffusion of NaNO₂out of the microparticles and therefore slows the rate of bubbleformation, which initially takes place on the microparticle surfaces. Inturn, this slower bubble formation provides for an extended period ofcontact between gaseous NO in the bubbles and the liquid, increasing thefraction of NO that dissolves into the liquid. In fact, if the releaseof NO from the microparticle is slow enough, the release rate of NO maybecome equal to or smaller than the rate of NO dissolution in theliquid, no free bubbles will be released into the liquid, and all the NOwill be dissolved. Thus, since the higher HEC concentration reduces theproduction rate of NO, a larger fraction of the NO will be dissolved inthe higher-HEC-concentration solution.

Another factor that increases the amount of NO dissolved in thehigher-HEC-concentration solution is that if the NO bubbles leave thesurfaces of the microparticles and become free bubbles of NO, theyreside in the liquid for much longer times because the higher-HECconcentration solution is a considerably more viscous gel than thelower-HEC concentration solution. This slow transport of free NO bubblesprovides an additional opportunity for additional NO to become dissolvedin the liquid.

The kinetic effects described above are more pronounced at higherconcentrations of HEC. Therefore, in the higher-concentration HECsolution, both because of the reduced rate of NO formation, and theconsequent improvements in transfer of NO to the liquid, thehigher-HEC-concentration solution develops a very significantly higherconcentration of NO, and maintains a high concentration of NO for asubstantial period of time, as shown in FIG. 6.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. An extended release generator of therapeuticnitric oxide comprising a mixture of two or more sub-millimeter-scaleparticles of separately microencapsulated reactants including particlesof at least one microencapsulated nitrite salt, particles of at leastone microencapsulated acid, and particles of at least onemicroencapsulated reducing agent, wherein the particles of separatelymicroencapsulated reactants are present in sufficient quantities toreact to produce NO when the microencapsulated reactants are slowlyreleased by addition of a sufficient volume of an aqueous solution toincipiently wet the sub-millimeter-scale particles.
 2. The extendedrelease generator of therapeutic nitric oxide of claim 1, wherein thenitrite salt is selected from one or more of the group consisting of:sodium nitrite, potassium nitrite, calcium nitrite, alkali metal nitriteand alkaline earth nitrite.
 3. The extended release generator oftherapeutic nitric oxide of claim 1, wherein the acid is an organic acidselected from one or more of the group consisting of: citric acid,lactic acid, glyceric acid, formic acid and ascorbic acid.
 4. Theextended release generator of therapeutic nitric oxide of claim 1,wherein the acid is boric acid.
 5. The extended release generator oftherapeutic nitric oxide of claim 1, wherein the reducing agent isselected one or more of the group consisting of: ascorbic acid, ascorbicacid derivatives, ascorbate salts, tocopherols, erythrobates,carotenoids, tocotrienols and thiols.
 6. The extended release generatorof therapeutic nitric oxide of claim 1, wherein the acid is citric acidand the reducing agent is ascorbic acid.
 7. The extended releasegenerator of therapeutic nitric oxide of claim 1, wherein the reactantsare encapsulated in a polymer selected from one or more of the groupconsisting of: ethyl cellulose, zein, chitosan, hyaluronic acid, alginicacid, biodegradable polyesters, polyanhydrides, poly (ortho esters),polyphosphazenes, and polysaccharides.
 8. The extended release generatorof therapeutic nitric oxide of claim 1, wherein a microencapsulatedascorbic acid or a microencapsulated ascorbic acid derivative functionsas both the microencapsulated acid and the microencapsulated reducingagent.
 9. The extended release generator of therapeutic nitric oxide ofclaim 1, wherein the extended release generator is disposed in a layeredpad comprising a mixture of the sub-millimeter-scale microencapsulatedreactants.
 10. The extended release generator of therapeutic nitricoxide of claim 9, wherein the layered pad comprises an outward-facingimpermeable layer and a bodyside-facing permeable layer and sandwichedtherebetween a mixture of the sub-millimeter-scale microencapsulatedreactants.
 11. The extended release generator of therapeutic nitricoxide of claim 9, wherein the layered pad is adapted and dimensioned asa wound dressing or bandage.
 12. The extended release generator oftherapeutic nitric oxide of claim 9, wherein the layered pad is adaptedand dimensioned as to be inserted into or manufactured as a part ofsocks, gloves, hats and other garments.
 13. The extended releasegenerator of therapeutic nitric oxide of claim 9, wherein the layeredpad is adapted and dimensioned as a bed pad.
 14. The extended releasegenerator of therapeutic nitric oxide of claim 9, wherein the layeredpad is adapted and dimensioned as an antimicrobial wipe cloth.
 15. Theextended release generator of therapeutic nitric oxide of claim 1,wherein the extended release generator is disposed in a condom.
 16. Amethod for treating a wound using the extended release generator oftherapeutic nitric oxide of claim
 1. 17. A method for treating hair lossusing the extended release generator of therapeutic nitric oxide ofclaim
 1. 18. A method for treating of sexual dysfunction using theextended release generator of therapeutic nitric oxide of claim
 1. 19. Amethod of providing extended release therapeutic nitric oxidecomprising: applying a mixture of sub-millimeter-scale microencapsulatedreactants including at least one microencapsulated nitrite salt, atleast one microencapsulated acid, and at least one microencapsulatedreducing agent; adding an activating volume of an aqueous solutionsufficient to incipiently wet the microencapsulated reactants andinitiate extended release production of NO via a nitrous acidintermediate.
 20. The method of claim 19, wherein the microencapsulatedacid and the microencapsulated reducing acid are together provided bymicroencapsulated ascorbic acid and/or a microencapsulated ascorbic acidderivative.
 21. The method of claim 19, wherein the microencapsulatedreactants are provided in a wound dressing or bandage.
 22. The method ofclaim 19, wherein the microencapsulated reactants are provided in a bedpad.
 23. The method of claim 19, wherein the microencapsulated reactantsare provided in a condom.
 24. A kit for providing extended releasetherapeutic nitric oxide to a patient comprising: a mixture ofsub-millimeter-scale microencapsulated reactants including at least onemicroencapsulated nitrite salt, at least one microencapsulated acid, andat least one microencapsulated reducing agent, an activating volume ofwater or an aqueous gel sufficient to admix with the microencapsulatedreactants, wherein the microencapsulated reactants and the aqueous gelare present in sufficient quantities to react to produce NO via anitrous acid intermediate when the microencapsulated reactants areslowly released by addition of the aqueous gel.
 25. The kit of claim 24,wherein the microencapsulated acid and the microencapsulated reducingagent are together provided by a microencapsulated ascorbic acid and/ora microencapsulated ascorbic acid derivative.
 26. The kit of claim 24,wherein the microencapsulated reactants are provided in a wound dressingor bandage and the activating volume of water or aqueous gel is providedin a premeasured container.
 27. The kit of claim 24, further comprisingan instruction to the patient to apply the mixture ofsub-millimeter-scale microencapsulated reactants to a tissue in needthereof and to add the activating volume of water or aqueous gel to thereactants
 28. The kit of claim 27, wherein the instructions furtherdirect the patient to add a reactivating volume of water or aqueous gelto the reactants after the reactants have dried.
 29. The kit of claim24, wherein the kit further includes at least one reactivating volume ofwater or aqueous gel in a premeasured container.